JP2002526755A - Raw inspection technology - Google Patents

Abstract

(57) Summary The present invention relates to a system for performing a parallel live test. For each biochemical test, a microfabricated label is prepared, and various kinds of labels are mixed with an analysis sample. The results of each test are separated by a device that reads each label. The micromarker has a surface layer of anodized metal and is manufactured by anodizing, patterning by lithography, and etching. Aluminum is the preferred metal.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention relates in particular, but not exclusively, to the field of micromachined or microfabricated coded substrates for use as a parallel biopsy technique.

[0002] a large amount and raw inspection to be carried out in parallel is a functional technologies that contribute to the development of the most modern in genetics, screening and drug discovery. The myriad tests previously performed one by one can now be reduced to a single experiment that produces myriad results. An important aspect of this method is the development of a technique that separates the results of many different tests from each other.

[0003] Some existing technologies are described below. The art consists of labeling each of the constituent experiments so that they can be read after the end of the experiment. Labels currently in use include the location of the experiment on the surface of the test chip and the fluorescence spectra of the particles that are relevant to the experiment.

The probe array of the GeneChip of Affymetrix is a chip for inspecting a DNA sequence, and a very large number of different DNA probes are arranged at predetermined points on a large two-dimensional array. Methods for making this are described, for example, in US 5,744,305 or US 5,143,854. Standard DNA hybridization techniques are used in a chamber on the chip, and the test results are read optically by the location of the fluorescent spots on the array (see, eg, US Pat. No. 5,578,832). The test combination is predetermined during manufacture of the chip.

[0005] Luminex Corporation's FlowMetrix system uses encoded microspheres with a diameter of 6.5 μm. Each bead incorporates red and orange fluorophores to form a code. Eight different strengths are possible, thus allowing 64 bead types. For the probe, a green fluorescent marker is used. This system is US 5,736,330
It is described in. This system has a relatively small number of codes and requires complex multi-wavelength optics on the flow cytometer to read the codes and the fluorescence of the specimen.

[0006] NanoGen has a semiconductor-based microchip array, APEX, which is
Intended for NA binding and sequencing experiments. Nan with various arrays of DNA probes
oGen chips can be programmed by end users (e.g., US 5,605,662 and U.S.A.).
S 5,929,208).

[0007] In general combinatorial chemistry, there are a number of other particle or substrate based inspection techniques. For example, WO 96/24061 describes a test object library using a radio frequency identification tag. WO 97/32892 describes a composite support for use in substrates for combinatorial chemistry. GB 2306484 describes bipartite support particles for combinatorial chemistry.

SUMMARY OF THE INVENTION [0008] The present invention relates to a system for performing low-cost, rapid and simple multiple biopsy tests performed in large numbers and in parallel. The system involves creating a suspension (test solution) containing a very large number of different types of microfabricated encoded labels (microlabels). Each encoded label carries a different biochemical reagent or probe.

Test solutions are made by mixing together a selected set of suspensions of active microlabels with each other. The test solution is made for a particular use, independent of the original fabrication of the microlabel.

The sample to be tested is marked with a fluorescent label and it is incubated with a test solution. Only the microlabel with a probe that binds to the fluorescent sample molecule emits fluorescence.

The micro-labels are made from a material that can be anodized, for example, aluminum.
First, it is deposited on a flat substrate with a soluble release layer. Before patterning,
The metal surface is anodized. This allows for the attachment of a wide range of biochemically active agents used as highly selective probes.

[0012] Individual micro-labels are obtained by patterning the aluminum using standard optical lithography and dry etching. The code is stored on the micro-marker as a series of holes by an encoding method similar to the bar code method. Biochemical probes can be attached to the surface either before or after the lithographic process. Next, the micro-labeled substance is peeled into the aqueous suspension. Up to 100,000 types of microlabels have been demonstrated.

A flow-based reading system, similar to a flow cytometer, passes through thousands of microlabels per second and reads both its barcode and test results. The test results are measured by the degree of fluorescence. Another plate reading system uses fluorescence microscopy and image processing to read test results. In this system, a small marker is placed on a flat substrate. Embodiments of the present invention will be described with reference to the drawings.

DETAILED DESCRIPTION fine labels Figure 1 shows made from aluminum, in the form of small optical barcode microfabricated micro labels 1. This barcode is formed by a series of holes 2 in aluminum.

Each of the micro-labels of this type has a length of about 100 μm (3), a width of 10 μm (4) and a thickness of 1 μm (5), and can store up to 100,000 kinds of codes. Single diameter 1
Approximately 10 million micromarkers can be produced on a 5.24 cm (6 inch) substrate. Microlabels of different lengths of 40-100 μm with 2-5 decimal digits of data are produced. For each different code, only one biochemical probe is linked. To perform a strong error test when reading that code, coding methods such as those used for EAN and UPC barcodes are used.

Probe and Test Solution Referring to FIG. 2, a test solution 6 of microlabels is made by mixing together a selected set of suspensions of active microlabels. Each different code has a unique biochemical probe attached to it, which binds to a particular type of molecule.
The binding reaction is selected from the group consisting of an antibody-antigen, an enzyme-substrate, an enzyme-receptor, a toxin-receptor, a protein-protein, and avidin-biotin.

The test sample 8 is marked with a fluorescent label and it is incubated with the test solution. Only the microlabeled body 7 having a probe that binds to the fluorescent sample molecule emits fluorescence 10. The test results are obtained by measuring the fluorescence intensity of different types of microlabels.

One example of the use of the present invention is to screen sera by parallel biopsy to select specific antibodies. The antigen for the antibody is used as a probe to bind to the marked microlabel for identifying the antigen. This microlabel is incubated with the sample and then with the antibody-specific fluorescent label. Next, the microlabel is passed through a reader that measures the fluorescence intensity of the label and identifies the label. The correlation between the antibody in the sample and the surface-bound antigen is displayed from the correlation between the identification of the microlabel and the fluorescence intensity.

Fabrication of a micro-marker A method of manufacturing a micro-marker will be described with reference to FIG. A substrate material 11, for example a silicon wafer, is first coated with a soluble release layer 12. In a preferred embodiment, this is a layer of spin-coated polymethyl methacrylate resist baked at 150 ° C. to remove solvents.

A 1 μm thick aluminum layer 13 is deposited on the substrate. This is performed by a standard vacuum sputter coating method generally used for manufacturing semiconductor devices.

The aluminum layer 13 is anodized 14 in a 4% phosphoric acid bath at a voltage of 30 V for 30 seconds at a current density of 10 mA / cm ² using a pure aluminum cathode. This forms a 100 nm anodized layer 15 having pores of about 40 nm in size.

At this stage (depending on the application), the aluminum surface may be coated with probe molecules 16 via dipping the surface in an aqueous solution / suspension of the molecule.

This substrate is spin-coated with normal optical resist 17, Shipley S1813. The marker pattern 18 is exposed using a hard-contact optical mask and developed with an aqueous alkaline developer (MF319). The pattern is transferred to the aluminum layer by a reactive ion etching method using SiCl ₄ .

If probe bonding is performed after the lithography process, the optical resist 17 may be removed at this stage with a solvent, for example, isopropyl alcohol. This results in the release layer 13 and the patterned aluminum layer 20 thereon.
Both remain complete. The probe molecules 16 can then be attached as before, while the microlabel is still attached to the substrate.

The micro-labeled body to which the probe has been adhered is separated from the substrate with a solvent such as acetone. By dilution and filtration, the microlabels 22 are made into an aqueous suspension that is immediately mixed with the test solution.

This fabrication method is suitable for many types of microlabels with holes, for example, the substantially linear ones of FIG. 1 as well as square, rectangular and circular microlabels.

Anodizing and surface chemistry When incubated in aqueous solutions, proteins bind only weakly to untreated aluminum surfaces. By modifying the surface, the time at which this binding occurs can be controlled and the binding can be selectively enhanced. This is important. This is because during manufacture, the probe molecules are strongly bound to the surface, but the non-specific binding of the fluorescent target molecule is maintained weak during the test. In this way, the identification of the specimen is maximized.

There is very extensive information available in the first half of this century on the surface protection of aluminum components by electrochemical methods. Methods for growing porous surfaces are well known, as are methods for sealing said surfaces. We have used this information to develop a relatively simple method of growing adsorbent surfaces with well-controlled porosity and depth. The surface binds the selected protein well shortly after treatment, but recovers over time to prevent further binding.

In order to form a suitable anodized surface morphology, it is necessary to understand the typical structure of a biomolecule used as a probe. Such biomolecules can be directly imaged by cryogenic atomic force microscopy. For example, immunoglobulin G (IgG)
Has a Y-shaped structure. The size of an IgG molecule spans about 40 nm, which can be considered a typical size.

As shown in FIG. 4, during anodic oxidation, an aluminum oxide layer 23 grows on the aluminum surface. This oxide layer is grown in a two-dimensional hexagonal cell structure 24. The size of each cell depends on the electrochemical formation voltage. At the center of each cell, a pore 25 having a diameter smaller than the width of each cell is formed. As the anodization process proceeds, the thickness 27 of this oxide layer and the depth 28 of the hole increase, but the cell width 26 and the hole diameter 28 remain constant.

Reading System Two reading systems have been developed. These are based on flow cytometry and on planar imaging of flattened microlabels.

As shown in FIG. 5, a flow cytometry-type reader is a micro-labeled substance 1
Is designed to flow down the center of the tube 29 and down to a defined reading zone 30. Normally, elongated particles in flow tend to roll around. However, by injecting the micro-label into the center 32 of its flow using the accelerating sheath fluid 31, a hydrodynamic focusing effect is obtained, so that the micro-label is aligned and the injection point 32 It passes through a well-defined focal point 30 that exists downstream.

At the hydrodynamic focus 30, two orthogonal focused laser light beams 33, 34 from a 488 nm argon ion laser are illuminated on the micro-label.
This is shown in detail in FIG. The use of two orthogonal irradiation beams allows irradiation of the micro-label, regardless of the rotation of the flow tube relative to the reference frame. In FIG. 6, micromarker 1 shows a 45 degree angle for both incident beams. this is,
This is the worst case possible. Nevertheless, it can be seen from the outline of the micromarker shown in FIG. 1 that the light still passes through the holes 2 of the marker. As the marker passes through, the hole modulates the transmitted intensity, resulting in a series of continuous information that is analyzed at the detector 35 in the same manner as a normal bar code.
At the same time, the fluorescence intensity is measured by the outer (epi-) fluorescence detector 36. This is correlated with the code on the label. To achieve the desired resolution, a high aperture optical element (microscope objective) is used. The optimal flow cytometric design has flat walls, eliminating the need for custom cylindrical imaging elements.

After reading a sufficient number of microlabels, the reader calculates all test results. The number required to perform statistical analysis is typically 10-100 copies per type of microlabel. The test results show the average value and standard deviation of fluorescence for each type of probe.

In the flat-plate reading system, the minute marker is arranged on a flat substrate. This substrate is either a filter substrate or a permeable substrate. An ordinary fluorescence microscope is used to systematically analyze the arrangement substrate. Each field image on the substrate is captured by the image processing system. The transmitted light is analyzed separately from the fluorescence. Each microlabel is identified by its transmitted light profile, while the integrated fluorescent signal is recorded on the surface of the label. Again, 10-100 copies of each type of microlabel are required for good statistical analysis.

[0036] In use typical use, using a number of different antibodies (e.g. hepatitis, HIV) suspension of probes for. In a reading system, a drop of blood is taken from the patient, labeled with a fluorescent marker, and incubated with the probe suspension for a few minutes. This suspension is supplied to a microfluidic detector system.

Other Embodiments The design of the microlabel for the flat plate reading system is not limited to the linear design described above, which is mainly used for a flow-through type reading system. Plate systems can use two-dimensional patterns fabricated on a sign that is closer to a square or rectangle than to a line. An encoding method similar to a normal two-dimensional barcode is used. The outer shape of the marker also provides information, especially regarding the direction.

[0038] A magnetic substance-incorporated label can be subjected to a magnetic separation method. This is useful if the sample to be tested contains a solid substance of a size close to the label, as this can confuse the results.

The microlabels can be made using any substrate that can be coated with a metal that can be anodized, eg, aluminum. This is particularly attractive when using high-volume manufacturing methods, such as, for example, embossing aluminum-coated plastics to make microlabels.

[Brief description of the drawings]

FIG. 1 shows a single microlabel incorporating a transmissive optical barcode.

FIG. 2 shows a parallel live test comprising two binding experiments.

FIG. 3 shows a method for producing a micro-label on a wafer scale.

FIG. 4 shows a porous structure formed by anodizing an aluminum surface.

FIG. 5 shows the flow of a micro-label in a reader.

FIG. 6 illustrates the use of orthogonal illumination beams to read a marker regardless of rotation.

[Procedure amendment]

[Submission date] March 29, 2001 (2001.3.29)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C25D 11/04 303 C25D 11/04 303 311 311 G01N 1/28 G01N 33/566 33/566 G06K 7/00 U G06K 7/00 G01N 37/00 102 // G01N 37/00 102 103 103 ZCC ZCC 1/28 N (72) Inventor, England, James United Kingdom, Cambridgeshire CB4 46 DG, Cambridge, Milton, Bat Lane 44A (72) Inventor Colby, Edward UK, Cambridgeshire, CB13 33 RL, Cambridge, Ireland Road 45 F-term (reference) 2G052 AA28 AD26 DA01 DA21 EC14 EC16 FD00 FD06 FD09 HB03 JA05 4G075 AA2 4 BC02 BC06 BD14 CA36 FB02 FC20 5B072 BB00 CC01 CC24 DD03 MM11

Claims (25)

[Claims] 1. A solid support for biochemical (binding experiments) testing, having an anodized metal surface layer. 2. The support according to claim 1, wherein said metal surface layer is aluminum. 3. Modified to enhance the affinity of the reagents of the binding experiment.
The support according to claim 1. 4. The method of manufacturing a support according to claim 1, wherein the flat surface is sputter-coated with the metal constituting the support, the metal is patterned by lithography, and each support is formed. Etching the metal to leave. 5. The method of claim 4, wherein said surface comprises a layer of a soluble material on a rigid support, such that said support is exfoliated by solvation of said soluble material. 6. The method according to claim 5, wherein said soluble material is a resist. 7. The support of claim 2, wherein the modification forms a porous surface. 8. The support according to claim 7, wherein the size of the pores is approximately equal to the size of the biochemically active molecule to be bound. 9. The method of making a support according to claim 3, wherein the modification is by an electrochemical method. 10. The method according to claim 9, wherein the modification is performed by anodizing the surface of the support.
The method described in. 11. The method according to claim 10, wherein said anodizing is performed at a voltage of up to 150 V.
The method described in. 12. The method of claim 11, wherein said anodizing is performed at a voltage of 4V to 30V. 13. The support preferably has a length of 100 μm, a width of 100 μm and a thickness.
The support according to claim 1, which is smaller than 100 µm. 14. The support of claim 1, wherein the support incorporates a spatially varying pattern for identification. 15. An optical reader for reading the pattern and identifying a support according to claim 14. 16. The support according to claim 14, wherein the pattern is a barcode. 17. The support according to claim 16, wherein the barcode is a linear barcode. 18. The reader according to claim 15, wherein the reader is operated by a transmission optical element. 19. The reader of claim 18, wherein the support is transported by a flow system into an optical reader. 20. The result of a number of binding experiments, according to the identification of the substrate according to claim 14, wherein one of the members of the binding pair is adhered as measured by the reader according to claim 15. How to separate. 21. The binding pair may be an antibody-antigen, an enzyme-substrate, an enzyme-receptor,
21. The method of claim 20, wherein the method is selected from the group consisting of a toxin-receptor, a protein-protein, and avidin-biotin. 22. The attached member of the binding pair is fluorescently labeled.
The method according to 0. 23. The pattern comprising a series of holes in the support.
The support according to claim 14. 24. The device of claim 18, having two substantially orthogonal light transmission paths.
Reader. 25. The reader according to claim 24, wherein the reader incorporates one or more fluorescence detectors.